A CELL-FREE ASSAY USING CYTOPLASMIC CELL-EXTRACTS TO STUDY REJOINING OF RADIATION-INDUCED DNA DOUBLE-STRAND BREAKS IN HUMAN CELL-NUCLEI

We describe a cell-free assay that can be employed to study rejoining of radiation-induced DNA double-strand breaks (dsbs) under in vitro conditions. The assay uses nuclei prepared from irradiated, agarose-embedded human A549 cells as substrate and cytoplasmic cell extracts prepared from exponentially growing HeLa cells as the source of enzymes. We demonstrate that rejoining of dsbs is absolutely dependent on cell extract and that, under optimal reaction conditions, it proceeds to an extent similar to that observed in intact cells, albeit with about six times longer half time. Dsb rejoining in this assay requires Mg2+ and is inhibited by high concentrations of either K+ or Na+. The assay should provide means for the biochemical characterization of the enzymology of eukaryotic cell DNA repair under conditions that retain chromatin structure. The assay can also be adapted to study repair of other types of damage induced in the DNA by ionizing or non-ionizing radiations, as well as by diverse chemical agents.